Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Representative confocal microscopy images of 14 and 24 DIV rat primary cortical neurons treated with 1 µM BODIPY-cholesterol (green) for 24 h and stained with antibodies against an early endosomal marker (EEA1, red), which stains endosomal compartments where internalized material ends up first, or a late endosomal/lysosomal marker (LIMP-I, red), which stains a mature state of endosomal compartments. 24 DIV neurons accumulate BODIPY-cholesterol in early and late-compartments (white arrows). (B) The plot compares the relative amount (% versus 12–14 DIV) of BODIPY-cholesterol taken by 12–14 DIV and 23–28 DIV primary cortical neurons in a 24 h period, calculated by the intracellular fluorescent signal (Ext 495 nm/Em 507) of BODIPY-cholesterol (nmol of cholesterol where from a BODIPY-cholesterol standard curve) and normalized by the total protein in the sample (% versus 12–14 DIV; n = 9 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0046). (A, C) Plot showing the percentage of the total EEA1-positive (n = 1,536) or LIMP-I positive (n = 1,269) compartments that accumulate BODIPY-cholesterol, calculated from confocal images as the ones shown in panel (A) (N = 3 independent experiments). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whiitney Test (**** P < 0.0001). (D) Western blot analysis of total lysates of 12–14 DIV, 18–21 DIV, and 23–28 DIV rat cortical neurons shows a pronounced decrease in NPC1 protein levels in 18–21 DIV neurons that is maintained in 23–28 DIV neurons. An antibody against beta-actin was used to normalize the protein levels. Below, a plot with the quantification of the bands of Western blot experiments as the one shown. Protein levels are relative to 12–14 DIV. The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.0001). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunnet’s multiple comparisons tests (n = 6 for 12–14 DIV and 18–21 DIV; n = 5 for 23–28 DIV, *** P < 0.001; P -value for 18–21 DIV to 12–14 DIV comparison: 0.0002; P -value for 23–28 DIV to 12–14 DIV comparison: 0.0004). (E) Western blot analysis of lysates of neuronal cultures at different aging points. The blot was tested with an antibody against the cholesterol transporter ABCA1 and actin. Below, a plot compares the relative ABCA1 protein levels between 12–14 DIV, 18–21 DIV and 23–28 DIV neuronal cultures. The bands corresponding to ABCA1 were quantified from blots such as the one shown above. Statistical significance was analyzed by Kruskal–Wallis Test ( P -value 0.0022). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunn’s Test (n = 9 independent lysates per group; ** P < 0.01; P -value for 23–28 DIV to 12–14 DIV comparison: 0.0036).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Confocal Microscopy, Staining, Marker, Two Tailed Test, Western Blot

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Representative transmission electron microscopy (TEM) images of multivesicular bodies (MVBs) from untreated (control) or 4 µg/μl U18666A-treated 14 DIV cortical neurons. Scale bars = 200 nm. (A, B) Plot showing the number of ILVs normalized by the area (nm 2 ) of the MVB that contains them, between untreated (control) or 4 µg/μl U18666A (U18)-treated 14 DIV neuronal cultures, quantified from TEM images such as the ones shown in panel (A) (n = 121 MVBs; N = 2 experiments). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann-Whitney Test (**** P < 0.0001). (C) The graph shows the quantification of extracellular vesicles by Nanosight from the medium of untreated 14 DIV neurons (control) or treated with increasing concentrations of U18666A. The graph shows single experiments, the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.009). Comparison of 4 µg/μl U18666A (U18)-treated neurons to control group was performed by Dunnet’s multiple comparisons test (** P < 0.001; n = 6 for control and 3 µg/μl; n = 5 for 2 and 4 µg/μl). (D) Western blot analysis of a total lysate of 14 DIV neuronal culture and total lysates of small extracellular vesicles isolated from the medium of 14 DIV untreated (Cnt) or U18-treated neurons. The blot was tested with an antibody against CD81, a marker for exosomes, and calnexin (an ER protein used as a negative marker for exosomes). (E) Western blot experiment of total lysates of 14 DIV neuronal cultures transduced with a lentivirus expressing a scrambled shRNA as a control (Cnt) or an shRNA against NPC1. Blots were probed with an antibody against NPC1 or actin as a loading control. (F) Representative TEM images of 14 DIV neuronal cultures transduced with a scrambled shRNA (shRNA-cnt) or an shRNA against NPC1 (shRNA-NPC1). (G) The plot compares the average number of ILVs normalized by the area (nm 2 ) of the MVB containing them, between shRNA-cnt and shRNA-NPC1 transduced neurons. (F) Data were obtained by quantification of TEM images as the ones shown in panel (F) (n = 100 MVBs for shRNA-cnt; n = 108 for shRNA-NPC1; N = 2 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whitney test (**** P < 0.0001).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Transmission Assay, Electron Microscopy, Two Tailed Test, MANN-WHITNEY, Western Blot, Isolation, Marker, Transduction, Expressing, shRNA

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A, C, D) Representative transmission electron microscopy images of multivesicular bodies (MVBs) from non-infected 21 DIV neurons (control; subpanels [a and c]) or infected (NPC overexpression, subpanels [c and d]) with a Sindbis virus expressing GFP-tagged NPC1 (GFP-NPC1). (B) Western blot analysis of total lysates of 21 DIV control neurons (cnt) or overexpressing GFP-NPC1 (NPC1). Blots were tested with an antibody against NPC1 and Actin. (C) The plot compares the number of ILVs normalized by the area of the MVBs that contained them (nm 2 ), between control and GFP-NPC1-overexpressing (NPC1) neurons. (A) Quantifications were performed on transmission electron microscopy images such as the ones shown in panel (A) (n = 181 MVBs for control; n = 161 MVBs for NPC1; N = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whitney test (**** P < 0.0001).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Transmission Assay, Electron Microscopy, Infection, Over Expression, Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Western blot analysis of total lysates of neuronal cultures at different aging points (10, 13, 15 and 17 DIV). The blot was tested with antibodies against NPC1, Akt phosphorylated at serine 473 (pAKT), total Akt, the mTOR substrate S6 Kinase (S6K) phosphorylated at threonine 389 (pS6K), total S6K, and actin. (A, B) The plot compares the levels of NPC1 normalized to actin, during neuronal aging in vitro. The protein levels were quantified from the bands of Western blot experiments as the ones shown in panel (A) (n = 4 independent cultures). The graph shows the mean ± SEM. The means for 10, 13, 15 and 17 DIV were significantly different when compared by ANOVA ( P -value: 0.0219). Dunnet’s multiple comparisons tests were used for the post hoc analysis of the data (** P < 0.01; P -value for the 10 DIV to 17 DIV comparison: 0.0049). (C) Alterations of the AKT-mTORC1 pathway during neuronal aging in vitro. (A) The plots show changes in the levels of pAKT (normalized to total AKT; upper plot) and pS6K (normalized to total S6K; lower plot) at the same aging points of neuronal cultures as in panel (A). (A) The protein levels were quantified from the bands of Western blot experiments as the ones shown in panel (A) (n = 3 independent cultures). The graph shows the mean ± SEM. The means for 10, 13, and 15 DIV (for pAKT, upper blot) and the means for 10, 15 and 17 DIV (for pS6K, lower blot) were significantly different when compared by ANOVA ( P -value for pAKT: 0.0308; P -value for pS6K: 0.0320). Dunnet’s multiple comparisons tests was used for the post hoc analysis of data (* P < 0.05; P -value for 15 DIV to 10 DIV comparison in pAKT: 0.0199; P -value for 15 DIV to 10 DIV comparison in pS6K: 0.0362; P -value for 17 DIV to 10 DIV comparison in pS6K: 0.0370). (D) Western blot analysis of the total lysates of N2A cells untreated or treated with 130 nM insulin growth factor for 24 h. The blot was tested with antibodies against NPC1, pAKT (ser473) and actin. Below, a plot comparing the NPC1 levels between untreated and Insulin Growth Factor-treated N2A cells. Quantifications were carried out on Western blot experiments as the one shown above (n = 4 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05; P -value: 0.0231). (E) Western blot analysis of total lysates of 14 DIV neuronal cultures treated with DMSO (control) or 5 µM of the pan-Akt activator SC79 for 24 h. The blot was tested with antibodies against NPC1, pAKT (ser473), and actin. Below, a plot comparing the NPC1 levels between DMSO-treated and SC79-treated neurons. Quantifications were carried out on Western blot experiments as the one shown above (n = 5 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05; P -value: 0.027). (C, F) N2A cells treated with DMSO (control) or Akt inhibitor VIII for 48 h were analyzed as in panel (C) (n = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0049). (F, G) Lysates of 17 DIV cortical neurons treated with DMSO (control) or the selective Akt inhibitor MK-2206 (2 µM) for 48 h were analyzed by Western blot with the same antibodies as in panel (F). Below, a plot comparing the NPC1 levels between DMSO-treated and MK-2206-treated neurons (n = 3 independent cultures). Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0052). (H) Lysates from 17 DIV cortical neurons treated with DMSO (control) or the mTOR inhibitor rapamycin (2.5 µM) were analyzed by Western blot. The blot was tested with antibodies against NPC1, pS6K (thr389), and actin. Below, the plot compares the NPC1 levels between DMSO-treated and 2.5 µM rapamycin-treated neurons. Quantification of the bands were carried out on blots such as the ones shown above (n = 3 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.01; P -value: 0.0053). (I) Comparison of the concentration (upper plot) and size (lower plot) of vesicles in the medium of 48 h DMSO (Cnt)- or 2.5 µl/ml rapamycin-treated 18 DIV neuronal cultures determined by Nanosight (n = 5 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05, ns: non-significant; P -value for concentration: 0.0134; P -value for size: 0.5072).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, In Vitro, Two Tailed Test, Concentration Assay

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) 14 DIV cortical neurons were treated with the NPC1 inhibitor U18666A (2 μg/ml) for 24 h or left untreated. Western blot analysis of total lysates show decrease mTOR-dependent phosphorylation of its substrate S6K. Actin was used to normalize for total protein. (B) The plot compares the levels of S6K phosphorylation between untreated (Control) or U18666A-treated (U18) neurons. (A) Quantification of pS6K levels from Western blot experiments as the one shown in panel (A) (n = 3). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) The plot compares the miR33 levels (normalized by the housekeeping RNA U6) between 13 DIV, 16–17 DIV and 21 DIV neuronal cultures (fold change versus DIV). The microRNA levels were determined by RT–PCR (n = 5 independent culture). The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA ( P -value: 0.0410). Dunnet’s multiple comparisons test was used to compare the 21 DIV and the 16–17 DIV to the 13 DIV group (ns: non-significant; * P < 0.05; P -value for the 21 DIV to 13 DIV comparison: 0.0258). (B) Western blot analysis of lysates of N2A cells transfected with 40 nM of a non-targeting control mimic or 40 nM miR-33 mimic (miRNA-33). The blots were analyzed with antibodies against NPC1, ABCA1 and actin. (C) Plot comparing the relative protein levels of NPC1 between N2A cells transfected with 40 nM of a non-targeting control mimic (n = 4) or 40 nM miR33 mimic (miR-33; n = 4). (B) Blots were quantified from Western blot experiments as the one shown in panel (B). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.05). (C, D) The plot compares the ABCA1 protein levels in the same experimental conditions as in panel (C). The graph shows the mean ± SEM and individual data points (n = 3). Statistical significance was analyzed by two-tailed unpaired t test (** P < 0.05). (E) Western blot analysis of brain lysates from 2 mo old WT or miR33 KO mice. Blots were tested with antibodies against NPC1 and HSP90. (F) Plots comparing the relative protein levels for NPC1 in the brain of WT (n = 5) and miR33 KO (n = 7) mice. (C) Protein levels were quantified from Western blot experiments as the one shown in panel (C). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (* P < 0.05).

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (A) Western blot analysis of brain lysates from 2 mo old WT or miR33 KO mice (same animals as in ). Blots were tested with antibodies against ABCA1 and HSP90 (same HSP90 blot as in ). (B) Plots comparing the relative protein levels for NPC1 in the brain of WT (n = 3) and miR33 KO (n = 3) mice. (A) Protein levels were quantified from Western blot experiments as the one shown in panel (A). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (*ns, non-significant). Although the statistical analysis did not reach significance, two of the WT mice show lower levels of ABCA1 in than the KO mice.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Western Blot, Two Tailed Test

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: (1) The activation of the AKT-mTOR pathway during neuronal aging triggers the degradation of the intracellular cholesterol transporter Niemann Pick disease, type C protein (NPC1), what induces the accumulation of cholesterol in endosomal compartments. (2) This in turn induces multivesicular bodies (MVBs) to generate a higher number of intraluminal vesicles (ILVs, called exosomes when they are secreted) in their lumen. (3) Decreased levels of NPC1 provoke a low endosome-to-ER cholesterol transport, what triggers the activation of the Sterol regulatory element-binding proteins through the Golgi and its translocation to the nucleus. There, sterol regulatory element-binding protein activates the transcription of the microRNA miR-33 located in the intronic region of the Srebf2 gene, after binding its sterol response element (SRE). (4) miR-33 blocks the synthesis of NPC1 by targeting its mRNA, what aggravates the accumulation of endosomal cholesterol and the formation of ILVs.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques: Activation Assay, Binding Assay, Translocation Assay

Journal: Life Science Alliance

Article Title: Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup

doi: 10.26508/lsa.202101055

Figure Lengend Snippet: Information on the antibodies used in this study.

Article Snippet: The plasmid containing EGFP-tagged NPC1 was purchased from Addgene (#53521).

Techniques:

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A) The mRNA levels of the indicated RABs in sh RABs MARC-145 cells were analyzed by qRT-PCR analysis. Values below the black line are statistically significance. (B) sh RABs MARC-145 cells were infected with PRRSV-2-GFP (MOI = 10) for 36 h. GFP-positive cells were analyzed by flow cytometry. Values above the green line (green bar) and below the red line (red bar) are statistically significance. (C) Immunoblot analysis of RAB18 and ACTB in scramble, sh RAB18 -1, and sh RAB18 -2 MARC-145 cells. (D) Scrambled, sh RAB18 -1, and sh RAB18 -2 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) and HP-PRRSV-2 (MOI = 10) for 36 h. Viral titers were assessed by the TCID 50 assay. *** P < 0.001. (E) Immunoblot analysis of RAB18 and ACTB in sgcontrol and sg RAB18 MARC-145 cells. (F) Sgcontrol and sg RAB18 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) and HP-PRRSV-2 (MOI = 10) for 36 h. Viral titers were assessed by the TCID 50 assay. *** P < 0.001. (G) MARC-145 cells were transfected with plasmids encoding RAB18-mCherry WT, RAB18-mCherry S22N, and RAB18-mCherry Q67L for 12 h and then infected with LP-PRRSV-2 (MOI = 10) for 36 h. PRRSV-2-N was detected by immunofluorescence analysis. Asterisks indicate non-transfected cells. Scale bar: 10 μm. (H) Quantification of the relative fluorescence intensity of PRRSV-2-N from (G) (n = 20). ** P < 0.01, *** P < 0.001. (I) Sgcontrol and sg RAB18 MARC-145 cells were transfected with plasmids encoding RAB18-FLAG WT, RAB18-FLAG S22N, and RAB18-FLAG Q67L for 12 h and then infected with LP-PRRSV-2 (MOI = 10) and HP-PRRSV-2 (MOI = 10) for 36 h. Viral titers were assessed by the TCID 50 assay. ** P < 0.01, *** P < 0.001. ns, not significance.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Quantitative RT-PCR, Infection, Flow Cytometry, Western Blot, Transfection, Immunofluorescence, Fluorescence

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A and B) PAM (A) and MARC-145 (B) cells were infected with LP-PRRSV-2 (MOI = 10) for 0–36 h. The mRNA levels of RAB18 were analyzed by qRT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001. (C and D) MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10, C) and HP-PRRSV-2 (MOI = 10, D) for 0–36 h. RAB18 and ACTB were analyzed by immunoblot analysis. (E) Immunohistochemistry for RAB18 and PRRSV-2-N in mock-infected or LP-PRRSV-2-infected porcine lungs (left). Asterisks indicate non-infected cells. Scale bar: 200 μm. The quantification of relative fluorescence intensity of PRRSV-2-N is shown on the right (n = 20). *** P < 0.001. (F) MARC-145 cells were transfected with the indicated RAB18-LUC plasmids for 12 h and then mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 24 h. RAB18 promoter activities were assessed by dual-luciferase reporter assay. *** P < 0.001. ns, not significance. (G) MARC-145 cells were transfected with sicontrol or si RELA -1 for 24 h, followed by infection with LP-PRRSV-2 (MOI = 10) for 24 h. ChIP assays were performed with IgG or an antibody against RELA. The RAB18 promoter was amplified by primer-1 (146 bp) and primer-2 (96 bp). (H) The treatment of MARC-145 cells was as described in (G). ChIP assays were performed with IgG or an antibody against RELA. *** P < 0.001. (I) MARC-145 cells were transfected with sicontrol, siR IG-I -1, si RIG-I -2, si MAVS -1, si MAVS -2, si RELA -1, si RELA -2, si RELB -1, and si RELB -2 for 36 h. The mRNA levels of RIG-I, MAVS, RELA, and RELB were analyzed by qRT-PCR analysis. *** P < 0.001. (J) MARC-145 cells were transfected with sicontrol, siR IG-I -1, si RIG-I -2, si MAVS -1, si MAVS -2, si RELA -1, si RELA -2, si RELB -1, and si RELB -2 for 24 h, then infected with LP-PRRSV-2 (MOI = 10) for 0–48 h. The mRNA levels of RAB18 were analyzed by qRT-PCR analysis. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Fluorescence, Transfection, Luciferase, Reporter Assay, Amplification

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A) PAM, scramble and sh RAB18 -1 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 0–24 h. LDs were analyzed by Oil Red O staining. Scale bar: 10 μm. (B) Quantification of LDs numbers per cell from (A) by ImageJ (n = 16). ** P < 0.01, *** P < 0.001. (C) Oil Red O staining of mock-infected or LP-PRRSV-2-infected porcine lungs. Scale bar: 200 μm. (D) Scramble and sh RAB18 -1 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 24 h. PRRSV-2-N was detected by immunofluorescence analysis and LDs were examined by BODIPY staining. Uninfected cells, indicated by asterisks, were outlined with dashed lines. Scale bar: 10 μm. (E) Quantification of relative BODIPY fluorescence intensity in cells from (D) by ImageJ (n = 16). ** P < 0.01. (F and G) PAM were infected with LP-PRRSV-2 (MOI = 10) for 0–24 h. TG (F) and NEFA (G) were quantified using biochemical kits. * P < 0.05, ** P < 0.01. (H and I) Scramble and sh RAB18 -1 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 0–24 h. TG (H) and NEFA (I) were quantified using biochemical kits. ** P < 0.01. ns, no significance. (J) Sgcontrol and sg RAB18 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 0–36 h. HSL, ATGL, PRRSV-2-N, RAB18, and ACTB were analyzed by immunoblot analysis. (K) Scramble and sh RAB18 -1 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 36 h. Viral assembly in the supernatants was determined by comparing the infectious titers (TCID 50 /mL) with the total PRRSV-2 genome equivalents (GE). ** P < 0.01.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Infection, Staining, Immunofluorescence, Fluorescence, Western Blot

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A) Sgcontrol and sg RAB18 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10) for 0–36 h. PLIN2, RAB18, PRRSV-2-N and ATCB were analyzed by immunoblot analysis. (B) MARC-145 cells were mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. Immunoblot analysis of PLIN2 (LDs), GM130 (Golgi), LAMP1 (Lysosome) and PRRSV-2-N was performed with cell lysates subjected to iodixanol density gradient centrifugation. (C) LDs were purified from MARC-145 cells mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. PLIN2, RAB18 and PRRSV-2-N were analyzed by immunoblot analysis. (D) Sgcontrol and sg RAB18 MARC-145 cells were transfected with a plasmid encoding GFP-RFP-PLIN2 for 12 h and then mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. PRRSV-2-N was detected by immunofluorescence analysis and the fluorescence of GFP and RFP was monitored by fluorescence microscopy. Scale bar: 10 μm. (E) Quantification of the fluorescence intensity ratio of RFP/GFP from D by ImageJ (n = 10). ** P < 0.01. ns, no significance. (F) MARC-145 cells were mock-infected or infected with LP-PRRSV-2 (MOI = 10), and treated with vehicle or CQ (10 mM) for 0–36 h. PLIN2, RAB18, PRRSV-2-N and ATCB were analyzed by immunoblot analysis.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Infection, Western Blot, Gradient Centrifugation, Purification, Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Microscopy

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A) Sgcontrol and sg RAB18 MARC-145 cells stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 2 h, and then mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. The fluorescence of KFERQ-PS-CFP2 was detected by fluorescence microscopy (left). Quantification of the KFERQ puncta per cell by ImageJ is shown on the right (n = 10). Scale bar: 10 μm. ** P < 0.01, *** P < 0.001. ns, no significance. (B) Sgcontrol and sg RAB18 MARC-145 cells were mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. PLIN2, LAMP2A, HSPA8, and NPC1 in lysosome purified by iodixanol density gradient centrifugation were analyzed by immunoblot analysis. (C) Immunoblot analysis of HSPA8 and ACTB in scramble and sh HSPA8 MARC-145 cells. (D and E) Scramble and sh HSPA8 MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10, D) and HP-PRRSV-2 (MOI = 10, E) for 36 h. Viral titers were assessed by the TCID 50 assay. ** P < 0.01. (F) Immunoblot analysis of LAMP2A and ACTB in scramble and sh LAMP2A MARC-145 cells. (G and H) Scramble and sh LAMP2A MARC-145 cells were infected with LP-PRRSV-2 (MOI = 10, G) and HP-PRRSV-2 (MOI = 10, H) for 36 h. Viral titers were assessed by the TCID 50 assay. ** P < 0.01.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Stable Transfection, Expressing, Infection, Fluorescence, Microscopy, Purification, Gradient Centrifugation, Western Blot

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: (A) MARC-145 cells were mock-infected or infected with LP-PRRSV-2 (MOI = 10) for 36 h. The interaction of RAB18, PLIN2, and HSPA8 was analyzed by Co-IP analysis with IgG (control) or anti-RAB18 antibody. (B) HEK293T cells were co-transfected with Rab18-HA and PLIN2-FLAG or PLIN2 (aa 1–251)-FLAG for 24 h. The interaction of RAB18 with PLIN2 variants was analyzed by Co-IP analysis. (C) HEK293T cells were co-transfected with HSPA8-HA and RAB18-FLAG for 24 h. The interaction of HSPA8 with RAB18 was analyzed by Co-IP analysis. (D) HEK293T cells were co-transfected with RAB18-FLAG and indicated HSPA8-HA variants for 24 h. The interaction of RAB18 with HSPA8 variants was analyzed by Co-IP analysis. (E) HEK293T cells were co-transfected with HSPA8-HA and indicated RAB18-FLAG variants for 24 h. The interaction of HSPA8 with RAB18 variants was analyzed by Co-IP analysis. (F) The interaction of His-SUMO-HSPA8 with indicated GST-RAB18 variants was assessed by in vitro affinity isolation assays. (G) The interaction of His-SUMO-RAB18 with indicated GST-HSPA8 variants was assessed by in vitro affinity isolation assays. (H) The interaction of His-SUMO-HSPA8 with GST-PLIN2 CTD was assessed by in vitro affinity isolation assays. (I) The interaction of His-SUMO-RAB18 with GST-PLIN2 CTD was assessed by in vitro affinity isolation assays.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Infection, Co-Immunoprecipitation Assay, Control, Transfection, In Vitro, Isolation

Journal: PLOS Pathogens

Article Title: Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18

doi: 10.1371/journal.ppat.1012123

Figure Lengend Snippet: PRRSV-2 infection activates the RIG-I/MAVS and canonical NF-κB pathways, which in turn upregulate RAB18 expression. RAB18 recruits PLIN2 through direct interaction with HSPA8 and thereby translocates PLIN2 to the lysosome for CMA-mediated degradation. Neutral lipases, such as ATGL, stimulate lipolysis, facilitating PRRSV-2 replication and assembly.

Article Snippet: The antibodies anti-RAB18 (11308-1-AP), anti-PLIN2 (15294-1-AP), anti-HSPA8 (10654-1-AP), and anti-NPC1 (13926-1-AP) were purchased from Proteintech; anti-LAMP1 (#9091), anti-GM130 (#12480), and anti-RELA (#3033) were purchased from Cell Signaling Technology; anti-LAMP2A (ab18528) was purchased from Abcam; anti-HA (A01244) and anti-His 6 (C157) were purchased from Genscript; anti-FLAG (F1804) and anti-ATCB (A1978) were purchased from Sigma-Aldrich; anti-PRRSV-2 nucleocapsid (N) (SDOW17) was purchased from Rural Technologies; horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (711-035-152) antibodies were purchased from Jackson ImmunoResearch Laboratories; anti-rabbit IgG antibodies labeled with Alexa Fluor 488 (A11034) and Alexa Fluor 568 (A11011) and anti-mouse IgG antibodies labeled with Alexa Fluor 488 (A11001), Alexa Fluor 568 (A11004), Alexa Fluor 405 (A31553), and Alexa Fluor Plus 647 (A32728) were purchased from Thermo Fisher Scientific.

Techniques: Infection, Expressing